The effect of acute resistance exercise on serum malondialdehyde in resistance-trained and untrained collegiate men

The purposes of this study were to determine whether acute resistance exercise increases serum malondialdehyde (MDA) levels postexercise, and if so, whether resistance exercise training status influences the magnitude of the exercise-induced lipid peroxidation response. Twelve recreationally resistance-trained (RT) and 12 untrained (UT) men who did not have resistance exercise experience in the past year participated in this study. All subjects completed an 8-exercise circuit resistance exercise protocol consisting of 3 sets of 10 repetitions at 10 repetitions maximum for each exercise. Blood samples were obtained pre-exercise, at 5 minutes postexercise, and at 6, 24, and 48 hours postexercise. At pre-exercise, MDA (nmol.ml(-1)) averaged 3.41 +/- 0.25 (RT) and 3.20 +/- 0.25 (UT) and did not differ (p > 0.05) either between groups or over time. Creatine kinase (IU.L(-1)) was significantly (p < 0.05) elevated 5 minutes postexercise (170.6 +/- 25.8), 6 hours postexercise (290.3 +/- 34.4), 24 hours postexercise (365.5 +/- 49.9), and 48 hours postexercise (247.5 +/- 38.5) as compared with pre-exercise (126.4 +/- 20.2) for both groups. There was no difference (p > 0.05) in CK activity between groups. This study indicated that moderate-intensity whole-body resistance exercise had no effect on serum MDA concentration in RT and UT subjects.     

Deconstructing the molecular portrait of basal-like breast cancer

Gene-expression profiling has revealed several molecular subtypes of breast cancer, which differ in their pathobiology and clinical outcomes. Basal-like tumors are a newly recognized subtype of breast cancer, which express genes that are characteristic of basal epithelial cells, such as the basal cytokeratins, and are associated with poor relapse-free and overall survival. However, the genetic and epigenetic alterations that are responsible for the biologically aggressive phenotype of these estrogen receptor-negative and HER2/ErbB2-negative tumors are not well understood, thereby hindering efforts to develop targeted therapies. Here, we focus on new insights into the molecular pathogenesis of basal-like breast cancer and explore how these discoveries might impact the treatment of these poor-prognosis tumors.     

Orthopedic gene therapy—lost in translation?

Orthopedic gene therapy has been the topic of considerable research for two decades. The preclinical data are impressive and many orthopedic conditions are well suited to genetic therapies. But there have been few clinical trials and no FDA-approved product exists. This paper examines why this is so. The reasons are multifactorial. Clinical translation is expensive and difficult to fund by traditional academic routes. Because gene therapy is viewed as unsafe and risky, it does not attract major funding from the pharmaceutical industry. Start-up companies are burdened by the complex intellectual property environment and difficulties in dealing with the technology transfer offices of major universities. Successful translation requires close interactions between scientists, clinicians and experts in regulatory and compliance issues. It is difficult to create such a favorable translational environment. Other promising fields of biological therapy have contemplated similar frustrations approximately 20 years after their founding, so there seem to be more general constraints on translation that are difficult to define. Gene therapy has noted some major clinical successes in recent years, and a sense of optimism is returning to the field. We hope that orthopedic applications will benefit collaterally from this upswing and move expeditiously into advanced clinical trials.     

Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.     

Prevention of hypothermic haloing extends the preservation time of hepatocytes at non freezing temperatures

Factors limiting the hypothermic preservation of hepatocytes on gelatin gels at 10 °C were investigated. Following 4 days of preservation, uniform morphological changes started to appear. The cells exhibited halos that increased in size. The particulate matter of the cell was confined to the central region. Cell viability was reduced from day 7 onwards. Neither fresh media changes nor the use of conditions to minimise free radical formation improved cell survival. However, haloing was decreased by short term temperature elevation to 37 °C (3 h), to reactivate the cells, and could be prevented completely by a stepwise increase in the sucrose concentration of the medium. The addition of sucrose in increments of 50 mM, at four day intervals, was found to inhibit morphological change. Prevention of haloing enabled the cells to be preserved for at least two weeks. The preserved cells attached to supports and spread as if freshly isolated. The procedure allows extended preservation of cells at non-freezing temperatures.     

Leaf growth characteristics of Vitis vinifera and prediction of final lamina length for studies of powdery mildew

Total functional leaf area is a key factor in determining crop yield. A nonlinear mixed‐effects model was employed to estimate growth responses for individual leaves using repeated measures of lamina length ≥30 mm, in the absence of disease. Resulting growth curves make allowances for, and allow assessment of, leaf to leaf variability. The major source of variability in leaf growth was identified as differences in thermal time required to reach half final lamina length. Juvenile leaves of Vitis vinifera are susceptible to infection by the powdery mildew fungus (Erysiphe necator) which impairs leaf function. The model was used to predict unobserved final lamina length for a subset of leaves inoculated with E. necator immediately after observations ceased. The severity of infection by E. necator varies among infected leaves. A previous study identified which of the inoculated leaves developed symptoms of severe powdery mildew. Maximum severity of infection was found to occur when individual leaves were at 85.3–97.9% of predicted final lamina length.     

Manipulating Connexin Communication Channels: Use of Peptidomimetics and the Translational Outputs

Gap junctions are key components underpinning multicellularity. They provide cell to cell channel pathways that enable direct intercellular communication and cellular coordination in tissues and organs. The channels are constructed of a family of connexin (Cx) membrane proteins. They oligomerize inside the cell, generating hemichannels (connexons) composed of six subunits arranged around a central channel. After transfer to the plasma membrane, arrays of Cx hemichannels (CxHcs) interact and couple with partners in neighboring attached cells to generate gap junctions. Cx channels have been studied using a range of technical approaches. Short peptides corresponding to sequences in the extra- and intracellular regions of Cxs were used first to generate epitope-specific antibodies that helped studies on the organization and functions of gap junctions. Subsequently, the peptides themselves, especially Gap26 and -27, mimetic peptides derived from each of the two extracellular loops of connexin43 (Cx43), a widely distributed Cx, have been extensively applied to block Cx channels and probe the biology of cell communication. The development of a further series of short peptides mimicking sequences in the intracellular loop, especially the extremity of the intracellular carboxyl tail of Cx43, followed. The primary inhibitory action of the peptidomimetics occurs at CxHcs located at unapposed regions of the cell’s plasma membrane, followed by inhibition of cell coupling occurring across gap junctions. CxHcs respond to a range of environmental conditions by increasing their open probability. Peptidomimetics provide a way to block the actions of CxHcs with some selectivity. Furthermore, they are increasingly applied to address the pathological consequences of a range of environmental stresses that are thought to influence Cx channel operation. Cx peptidomimetics show promise as candidates in developing new therapeutic approaches for containing and reversing damage inflicted on CxHcs, especially in hypoxia and ischemia in the heart and in brain functions.     

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4⁺ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.